Porcine reproductive and respiratory syndrome virus resistant animals

ABSTRACT

The present invention generally relates to genetically modified swine wherein at least one allele of a SIGLEC1 gene has been inactivated and/or at least one allele of a CD163 gene has been inactivated. Genetically modified swine having both alleles of the SIGLEC1 gene and/or both alleles CD163 gene inactivated are resistant to porcine reproductive and respiratory syndrome virus (PRRSV). Methods for producing such transgenic swine are also provided.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/117,943, filed Nov. 15, 2013, which is a U.S. national stageapplication of International Patent Application No. PCT/US2012/038193,filed May 16, 2012, which claims the benefit of U.S. ProvisionalApplication Ser. No. 61/519,076, filed May 16, 2011. Each of theabove-cited applications is incorporated by reference herein in itsentirety.

GOVERNMENT SUPPORT

This invention was made with Government support under the contractnumber (USDA/ARS) 58-1940-8-868 awarded by THE Department ofAgriculture. The Government has certain rights in the invention.

SEQUENCE LISTING

This application contains a Sequence Listing entitled “UMO 11053.WOSEQ₁₃ ST25” generated on May 15, 2012, which is hereby incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present invention generally relates to genetically modified swinewherein at least one allele of a SIGLEC1 gene has been inactivatedand/or at least one allele of a CD163 gene has been inactivated. Methodsfor producing such transgenic swine are also provided.

BACKGROUND OF THE INVENTION

Porcine Reproductive and Respiratory Syndrome (PRRS) is one of the mosteconomically important diseases of swine. This disease was firstdetected in the United States in 1987 (Keffaber 1989) and in Europe in1990 (Wensvoort et al. 1991). Molecular analysis of the prototype PRRSviruses (PRRSV) VR-2332 and Lelystad (U.S. and European isolates,respectively) has suggested that divergently evolved strains emerged ontwo continents almost simultaneously, perhaps due to similar changes inswine management practices (Murtaugh et al. 1995; Nelsen et al. 1999).Since its initial emergence, this virus has spread worldwide, and PRRSVof the European genotype has been detected in U.S. swine herds (Ropp etal. 2004). PRRS is characterized by severe and sometimes fatalrespiratory disease and reproductive failure, but also predisposesinfected pigs to bacterial pathogens as well as other viral pathogens(Benfield et al. 1992), and is a key component of the economicallysignificant Porcine Respiratory Disease Complex (PRDC). The mostconsistent pathological lesions caused by PRRSV during acute infectionare interstitial pneumonia and mild lymphocytic encephalitis (Plagemann1996). After the acute phase of PRRSV infection, which is typicallycharacterized by viremia and clinical disease, many pigs fully recoveryet carry a low-level viral infection for an extended period of time.These “carrier” pigs are persistently infected with PRRSV and shed thevirus, either intermittently or continuously, and may infect naïve pigsfollowing direct or indirect contact. Under experimental conditions,persistent infection with PRRSV has been well documented (Albina et al.1994; Allende et al. 2000; Benfield et al. 1998; Christopherhennings etal. 1995; Sur et al. 1996; Yoon et al. 1993). Most notably, infectiousvirus has been recovered for up to 157 days post-infection (Wills et al.1997). Tissue macrophages and monocytes are the major target cellsduring both acute and persistent infection (Molitor et al. 1997),although pneumocytes and epithelial germ cells of the testes have alsobeen shown to be infected (Sur et al. 1996; Sur et al. 1997).

The etiologic agent of PRRS is an enveloped, positive-stranded RNA virusthat is a member of the Arteriviridae family, order Nidovirales. Othermembers of the Arteriviridae include Lactate dehydrogenase-elevatingvirus (LDV) of mice, Equine arteritis virus (EAV), and Simianhemorrhagic fever virus (SHFV). Analysis of genomic sequence datareveals extensive diversity among strains of PRRSV but alsowell-conserved domains (Andreyev et al. 1997; Meng 2000; Meng et al.1995). The genome organization of PRRSV is similar to that of otherArteriviruses, with the genomic RNA functioning as the messenger RNA forthe ORF1a replicase proteins (Plagemann 1996). ORFs1a and 1b compriseapproximately 80% of the viral genome and encode the RNA-dependent RNApolymerase as well as polyproteins that are processed into othernonstructural proteins (Snijder and Meulenberg 1998). Using Lelystadvirus, ORFs 2-7 have been determined to encode the viral structuralproteins. The protein encoded by ORF 5 (GP5) and M (Van Breedam et al.2010b) may play a role in the induction of apoptosis by PRRSV (Suarez etal. 1996; Sur et al. 1997) and is thought to be the viral attachmentprotein in the closely related Lactate dehydrogenase-elevating virus.The minor envelope glycoproteins GP2a and GP4 of the PRRSV interact withCD163 (Das et al. 2010). There are data suggesting that binding toSIGLEC1 (sialic acid binding Ig-like lectin 1) is necessary for entryinto the cells, and in fact dual binding to both SIGLEC1 and CD163appears to be needed for viral infection (Van Gorp et al. 2008).

Many characteristics of both PRRSV pathogenesis (especially at themolecular level) and epizootiology are poorly understood thus makingcontrol efforts difficult. To gain a better understanding, infectiousclones for PRRSV have been developed (Nielsen et al. 2003). Todayproducers often vaccinate swine against PRRSV with modified-liveattenuated strains or killed virus vaccines. However, current vaccinesoften do not provide satisfactory protection, due to both the strainvariation and inadequate stimulation of the immune system. A protectiveimmune response is possible since it has been demonstrated that previousexposure can provide complete protection when pigs are challenged with ahomologous strain of PRRSV (Lager et al. 1999). However, protectiveimmunity has never been consistently demonstrated for challenge withheterologous strains. In addition to concerns about the efficacy of theavailable PRRSV vaccines, there is strong evidence that themodified-live vaccine currently in use can persist in individual pigsand swine herds and accumulate mutations (Mengeling et al. 1999), as hasbeen demonstrated with virulent field isolates following experimentalinfection of pigs (Rowland et al. 1999). Furthermore, it has been shownthat vaccine virus is shed in the semen of vaccinated boars(Christopherhennings et al. 1997). As an alternative to vaccination,some experts are advocating a “test and removal” strategy in breedingherds (Dee and Molitor 1998). Successful use of this strategy depends onremoval of all pigs that are either acutely or persistently infectedwith PRRSV, followed by strict controls to prevent reintroduction of thevirus. The difficulty, and much of the expense, associated with thisstrategy is that there is little known about the pathogenesis ofpersistent PRRSV infection and thus there are no reliable techniques toidentify persistently infected pigs.

A putative cellular receptor for PRRSV has been identified by monoclonalantibodies, purified and sequenced (Vanderheijden et al. 2003; Wissinket al. 2003) and named SIGLEC1. This molecule has similarity tosialoadhesins and was shown to mediate entry of PRRSV intonon-susceptible cells but a recombinant cell line expressing thisreceptor failed to support productive replication of PRRSV(Vanderheijden et al. 2003). Importantly, the sialic acid moleculespresent on the PRRSV surface have been shown to be needed for infectionof alveolar macrophages. Following viral binding to this receptor, entryof PRRSV occurs by receptor-mediated endocytosis (Nauwynck et al. 1999).The key role of sialoadhesin for PRRSV entry was established byexperiments in which viral uptake was demonstrated in PK15 cells (a cellline non-permissive for PRRSV replication) that were transfected withcloned porcine sialoadhesin, but not in un-transfected control PK15cells (Vanderheijden et al. 2003). Further research by this same groupdemonstrated that interactions between the sialic acid on the PRRSVvirion surface and the sialoadhesin molecule were essential for PRRSVinfection of alveolar macrophages (Delputte and Nauwynck 2004). Areverse strategy, i.e. removing the sialic acid on the surface of thePRRSV or preincubation with sialic acid-specific lectins also resultedin blocking of the infection (Delputte et al. 2004; Delputte andNauwynck 2004; Van Breedam et al. 2010b). Independent of the specificresearch on PRRSV entry, site-directed mutagenesis has been used toidentify six key amino acid residues required for sialic acid binding bymurine sialoadhesin (Vinson et al. 1996), which is 69% identical toporcine sialoadhesin. Importantly, all six amino acids identified inmurine sialoadhesin are also conserved in the porcine molecule.

SIGLEC1 is a transmembrane receptor of a family of sialic acid bindingimmunoglobulin-like lectins. It was first described as a sheeperythrocyte binding receptor of mouse macrophages (Crocker and Gordon1986). It is expressed on macrophages in hematopoietic and lymphoidtissues. SIGLECs consist of an N-terminal V-set domain containing thesialic acid binding site followed by a variable number of C2-setdomains, and then a transmembrane domain and a cytoplasmic tail. Incontrast to other SIGLECs, SIGLEC1 does not have a tyrosine-based motifin the cytoplasmic tail (Oetke et al. 2006). The sialic acid-bindingsite was mapped to the N-terminal immunoglobulin-like V-set domain (Nathet al. 1995). The R116 residue appears to be one of the important aminoacids for sialic acid binding (Crocker et al. 1999; Delputte et al.2007). Thus, an intact N-terminal domain is both necessary andsufficient for PRRSV binding to SIGLEC1 (Van Breedam et al. 2010a). Aknockout of SIGLEC1 was reported in mice and resulted in mice that wereviable and fertile and showed no developmental abnormalities (Oetke etal. 2006). However, these mice had subtle changes in B- and T-cellpopulations and a reduced immunoglobulin M level.

The infection process of the PRRSV begins with initial binding toheparan sulfate on the surface of the alveolar macrophage. Securebinding then occurs to sialoadhesin (SIGLEC1, also referred to as CD169or SN). The virus is then internalized via clatherin-mediatedendocytosis. Another molecule, CD163, then facilitates the uncoating ofthe virus in the endosome (Van Breedam et al. 2010a). The viral genomeis released and the cell infected.

CD163 has 17 exons and the protein is composed of an extracellularregion with 9 scavenger receptor cysteine-rich (SRCR) domains, atransmembrane segment, and a short cytoplasmic tail. Several differentvariants result from differential splicing of a single gene (Ritter etal. 1999a; Ritter et al. 1999b). Much of this variation is accounted forby the length of the cytoplasmic tail.

CD163 has a number of important functions, including acting as ahaptoglobin-hemoglobin scavenger receptor. Elimination of freehemoglobin in the blood is an important function of CD163 as the hemegroup can be very toxic (Kristiansen et al. 2001). CD163 has acytoplasmic tail that facilitates endocytosis. Mutation of this tailresults in decreased haptoglobin-hemoglobin complex uptake (Nielsen etal. 2006). Other functions of C163 include erythroblast adhesion(SRCR2), being a TWEAK receptor (SRCR1-4 & 6-9), a bacterial receptor(SRCR5), an African Swine Virus receptor (Sanchez-Torres et al. 2003),and a potential role as an immune-modulator (discussed in (Van Gorp etal. 2010a)).

SUMMARY OF THE INVENTION

In one aspect, the present invention is a genetically modified swinewherein at least one allele of a SIGLEC1 gene has been inactivatedand/or at least one allele of a CD163 gene has been inactivated, whereininactivation of the CD163 allele results in a CD163 protein which cannotbind and/or uncoat a porcine reproductive and respiratory syndrome virus(PRRSV).

Another aspect of the present invention is a genetically modified swinewherein at least one allele of a SIGLEC1 gene has been inactivated,produced by a method comprising enucleating a swine oocyte; fusing theoocyte with a donor swine fibroblast cell, the genome of the fibroblastcell comprising at least one inactivated SIGLEC1 allele; and activatingthe oocyte to produce an embryo.

The present invention also relates to a genetically modified swinewherein at least one allele of a CD163 gene has been inactivated,wherein inactivation of the CD163 allele results in a CD163 proteinwhich cannot bind and/or uncoat a porcine reproductive and respiratorysyndrome virus (PRRSV), produced by a method comprising enucleating aswine oocyte; fusing the oocyte with a donor swine fibroblast cell, thegenome of the fibroblast cell comprising at least one inactivated CD163allele; and activating the oocyte to produce an embryo.

In another aspect, the present invention is a genetically modified swinewherein both alleles of a SIGLEC1 gene have been inactivated, producedby a method comprising mating a male genetically modified swine whereinat least one allele of the SIGLEC1 gene has been inactivated with afemale genetically modified swine wherein at least one allele of theSIGLEC1 gene has been inactivated to produce F1 progeny, and screeningthe F1 progeny to identify genetically modified swine wherein bothalleles of the SIGLEC1 gene have been inactivated.

It is another aspect of the present invention to provide a geneticallymodified swine wherein both alleles of a CD163 gene have beeninactivated, produced by a method comprising mating a male geneticallymodified swine wherein at least one allele of the CD163 gene has beeninactivated with a female genetically modified swine wherein at leastone allele of the CD163 gene has been inactivated to produce F1 progeny,and screening the F1 progeny to identify genetically modified swinewherein both alleles of the CD163 gene have been inactivated.

The present invention also relates to a genetically modified swinewherein both alleles of a SIGLEC1 gene and both alleles of a CD163 genehave been inactivated, wherein inactivation of the CD163 allele resultsin a CD163 protein which cannot bind and/or uncoat a porcinereproductive and respiratory syndrome virus (PRRSV), which is producedby any of three methods. The first such method comprises mating agenetically modified swine having at least one inactivated SIGLEC1allele with a genetically modified swine having at least one inactivatedCD163 allele to produce F1 progeny, and screening the F1 progeny toidentify genetically modified swine wherein at least one allele of theSIGLEC1 gene has been inactivated and at least one allele of the CD163gene has been inactivated. This method further comprises mating thegenetically modified swine wherein at least one allele of the SIGLEC1gene has been inactivated and at least one allele of the CD163 gene hasbeen inactivated to each other to produce F2 progeny and screening theF2 progeny to identify genetically modified swine wherein both allelesof the SIGLEC1 gene and both alleles of the CD163 gene have beeninactivated.

The second such method comprises mating a genetically modified swinewherein both alleles of a SIGLEC1 gene have been inactivated with agenetically modified swine wherein both alleles of a CD163 gene havebeen inactivated to produce F1 progeny, mating the F1 progeny to produceF2 progeny, and screening the F2 progeny to identify geneticallymodified swine wherein both alleles of the SIGLEC1 gene and both allelesof the CD163 gene have been inactivated.

The third such method comprises mating a genetically modified swinewherein at least one allele of a SIGLEC1 gene and at least one allele ofa CD163 gene have been inactivated to another genetically modified swinewherein at least one allele of a SIGLEC1 gene and at least one allele ofa CD163 gene have been inactivated to produce F1 progeny, and screeningthe F1 progeny to identify genetically modified swine wherein bothalleles of the SIGLEC1 gene and both alleles of the CD163 gene have beeninactivated.

The present invention also relates to progeny any of the above-describedgenetically modified swine, wherein: (1) at least one allele of aSIGLEC1 gene has been inactivated; (2) at least wherein at least oneallele of a CD163 gene has been inactivated; (3) at least one allele ofa SIGLEC1 gene and at least one allele of a CD163 gene have beeninactivated; or (4) both alleles of a SIGLEC1 gene and both alleles of aCD163 gene have been inactivated. In such progeny wherein one or bothalleles of a CD163 gene have been inactivated, the inactivation ofresults in a CD163 protein which cannot bind and/or uncoat a porcinereproductive and respiratory syndrome virus (PRRSV).

The present invention is also directed to a method for producing agenetically modified swine wherein at least one allele of a SIGLEC1 genehas been inactivated. The method comprises enucleating a swine oocyte;fusing the oocyte with a donor swine fibroblast cell, the genome of thefibroblast cell comprising at least one inactivated SIGLEC1 allele; andactivating the oocyte to produce an embryo.

In still another aspect, the present invention is a method for producinga genetically modified swine wherein at least one allele of a CD163 genehas been inactivated, wherein inactivation of the CD163 allele resultsin a CD163 protein which cannot bind and/or uncoat a porcinereproductive and respiratory syndrome virus (PRRSV). This methodcomprises enucleating a swine oocyte; fusing the oocyte with a donorswine fibroblast cell, the genome of the fibroblast cell comprising atleast one inactivated CD163 allele; and activating the oocyte to producean embryo.

The present invention is also directed to a method for producing agenetically modified swine wherein both alleles of a SIGLEC1 gene havebeen inactivated. The method comprises mating a female geneticallymodified swine having at least one inactivated SIGLEC1 allele with amale genetically modified swine having at least one inactivated SIGLEC1allele to produce F1 progeny, and screening the F1 progeny to identifygenetically modified swine wherein both alleles of the SIGLEC1 gene havebeen inactivated.

The present invention is also directed to a method for producing agenetically modified swine wherein both alleles of a CD163 gene havebeen inactivated, wherein inactivation of the CD163 allele results in aCD163 protein which cannot bind and/or uncoat PRRSV. This methodcomprises mating a female genetically modified swine having at least oneinactivated CD163 allele with a male genetically modified swine havingat least one inactivated CD163 allele to produce F1 progeny, andscreening the F1 progeny to identify genetically modified swine whereinboth alleles of the CD163 gene have been inactivated.

Another aspect of the present invention is a method for producing agenetically modified swine wherein both alleles of a SIGLEC1 gene andboth alleles of a CD163 gene have been inactivated, wherein inactivationof the CD163 alleles results in a CD163 protein which cannot bind and/oruncoat a porcine reproductive and respiratory syndrome virus (PRRSV).The method comprises mating a genetically modified swine having at leastone inactivated SIGLEC1 allele with a genetically modified swine havingat least one inactivated CD163 allele to produce F1 progeny, andscreening the F1 progeny to identify genetically modified swine whereinat least one allele of the SIGLEC1 gene has been inactivated and atleast one allele of the CD163 gene has been inactivated. The methodfurther comprises mating the genetically modified swine wherein at leastone allele of the SIGLEC1 gene has been inactivated and at least oneallele of the CD163 gene has been inactivated to each other to produceF2 progeny and screening the F2 progeny to identify genetically modifiedswine wherein both alleles of the SIGLEC1 gene and both alleles of theCD163 gene have been inactivated.

The present invention also relates to another method for producing agenetically modified swine wherein both alleles of a SIGLEC1 gene andboth alleles of a CD163 gene have been inactivated, wherein inactivationof the CD163 gene results in a CD163 protein which cannot bind and/oruncoat a porcine reproductive and respiratory syndrome virus (PRRSV).This method comprises mating a genetically modified swine wherein bothalleles of a SIGLEC1 gene have been inactivated with a geneticallymodified swine wherein both alleles of a CD163 gene have beeninactivated to produce F1 progeny, mating the F1 progeny to produce F2progeny, and screening the F2 progeny to identify genetically modifiedswine wherein both alleles of the SIGLEC1 gene and both alleles of theCD163 gene have been inactivated.

The present invention is also directed to yet another method forproducing a genetically modified swine wherein both alleles of a SIGLEC1gene and both alleles of a CD163 gene have been inactivated, whereininactivation of the CD163 gene results in a CD163 protein which cannotbind and/or uncoat a porcine reproductive and respiratory syndrome virus(PRRSV). The method comprises mating a genetically modified swinewherein at least one allele of a SIGLEC1 gene and at least one allele ofa CD163 gene have been inactivated to another genetically modified swinewherein at least one allele of a SIGLEC1 gene and at least one allele ofa CD163 gene have been inactivated to produce F1 progeny, and screeningthe F1 progeny to identify genetically modified swine wherein bothalleles of the SIGLEC1 gene and both alleles of the CD163 gene have beeninactivated.

In other aspects, the present invention relates to progeny ofgenetically modified swine produced by any of the above methods, whereinone or both alleles of a SIGLEC1 gene have been inactivated and/orwherein one or both alleles of a CD163 gene have been inactivated,wherein inactivation of the CD163 alleles results in a CD163 proteinwhich cannot bind and/or uncoat a porcine reproductive and respiratorysyndrome virus (PRRSV).

Other objects and features will be in part apparent and in part pointedout hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the organization of the sialoadhesin gene and thetargeting vector design. Panels A and B of FIG. 1 are schematic diagramsshowing that the human (panel A) and mouse (panel B) sialoadhesin genesrespectively are composed of 21 exons and span approximately 20 kb.Panel C of FIG. 1 shows murine mutational analyses of exon 2 (the DNAsequence of exon 2 as shown in panel C of FIG. 1 is SEQ ID NO: 7, andthe amino acid sequence encoded by exon 2 as shown in panel C of FIG. 1is SEQ ID NO: 8). The mutational analysis revealed 6 amino acids thatconferred binding of sialoadhesin to ligand (shown in boxed/bold text).Panel D of FIG. 1 depicts a targeting vector design that was used toreplace part of exon 1 and exons 2 and 3 of SIGLEC1 with stop codons.The vector also included a neomycin (neo) selection cassette driven by aPGK promoter.

FIG. 2 depicts the targeting vector design, the organization of thesialoadhesin gene, and the organization of the altered sialoadhesingene.

FIG. 3 is a photograph of a gel showing the PCR screening to identifytargeting of the sialoadhesin gene.

FIG. 4 is a photograph of a gel showing the PCR screening to identifythe equal presence of both wild type and targeted sialoadhesin alleles.

FIG. 5 depicts the structural domain organization of wild-type CD163 (onthe left) containing 9 extracellular SRCR domains, 2 proline, serine,and threonine (PST)-rich domains, a transmembrane region and anintracellular cytoplasmic tail. The genetically modified CD163 isdepicted on the right. The structural domain organization remains thesame except that the SRCR domain 5 has been replaced with SRCR domain 8from CD163 ligand (CD163L).

FIG. 6 depicts the CD163 targeting vector, wherein the arms of thevector are sections of DNA that have an identical sequence with thenaturally occurring or wild-type CD163, thus allowing the vector toanneal to the CD163 already present in the cells. The modified DNA thatlies between the two arms of CD163 can then be inserted into the cells'DNA by homologous recombination.

DEFINITIONS

A “knockout swine” is a genetically modified swine in which the functionof one or both alleles of a gene has been altered, for example, bypartial or complete gene deletion. If one allele of a gene is knockedout, the swine is heterozygous for that gene knock-out; if both allelesare knocked out, the swine is homozygous for the gene knockout.

The term “donor cell” refers to a cell from which a nucleus or chromatinmaterial is derived, for use in nuclear transfer. As is discussedelsewhere herein, nuclear transfer can involve transfer of a nucleus orchromatin only as isolated from a donor cell, or transfer of an entiredonor cell including such a nucleus or chromatin material.

The term “genetic modification” refers to one or more alterations in agene sequence (including coding sequences and non-coding sequences, suchas introns, promoter sequences, and 5′ and 3′-untranslated sequences)that alter the expression or activity of the gene. Such modificationsinclude, for example, insertions (of, e.g., heterologous sequences, suchas selectable markers, and/or termination signals), deletions, frameshift mutations, nonsense mutations, missense mutations, pointmutations, or combinations thereof.

The term “recipient cell” refers to a cell into which a donor cell, adonor cell nucleus, or donor cell chromatin is introduced. Recipientcells are suitably enucleated prior to nuclear transfer. Examples ofrecipient cells include oocytes, zygotes, and the cells of two-cellembryos.

“Small interfering RNA” (siRNA) refers to double-stranded RNA moleculeswhich have the ability to specifically interfere with proteinexpression. siRNAs are generally from about 10 to about 30 nucleotidesin length. The length of the siRNA molecule is based on the length ofthe antisense strand of the siRNA molecule.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is directed to genetically modified swine whichare resistant to infection by porcine respiratory and reproductivesyndrome virus (PRRSV). PRRSV infectivity results from three specificentry mediators: (1) initial binding with heparan sulfate, (2)binding/internalization by sialoadhesin (SIGLEC1), and (3)internalization/uncoating of the virus by CD163. Thus, prevention ofinteraction between PRRSV and SIGLEC1 and/or PRRSV and CD163 results inthe inability of PRRSV to establish the infection in a host. As such,the present invention is directed to genetically modified swine, whereinat least one allele of a SIGLEC1 gene has been inactivated and/orwherein at least one allele of a CD163 gene has been inactivated,wherein inactivation of the CD163 allele results in a CD163 proteinwhich cannot bind and/or uncoat a porcine reproductive and respiratorysyndrome virus (PRRSV). These swine can also be referred to as swineknockouts for SIGLEC1 and/or CD163.

The invention includes swine in which only one allele of a targeted gene(SIGLEC1 and/or CD163) has been inactivated, while the other allele hasremained unaffected. These animals, which are referred to herein as“heterozygous” or “hemizygous” animals can be used in breedingapproaches to generate homozygous mutants. Also included in theinvention are homozygous mutant swine, in which both alleles of a targetgene are inactivated, either by the same or by different approaches.Accordingly, the present invention includes genetically modified swinewherein: (1) one allele of a SIGLEC1 gene has been inactivated; (2) oneallele of a CD163 gene has been inactivated; (3) both alleles of aSIGLEC1 gene have been inactivated; (4) both alleles of a CD163 genehave been inactivated; (5) both alleles of a SIGLEC1 gene and one alleleof a CD163 gene have been inactivated; (6) one allele of a SIGLEC1 geneand both alleles of a CD163 gene have been inactivated; (7) one alleleof a SIGLEC1 gene and one allele of a CD163 gene have been inactivated;or (8) both alleles of a SIGLEC1 gene and both alleles of a CD163 genehave been inactivated. In each of these instances and in the context ofthe present application generally, the inactivation of the CD163allele(s) results in a CD163 protein which cannot bind and/or uncoat aporcine reproductive and respiratory syndrome virus (PRRSV).

Gene targeting carried out to make the animals of the invention canresult in gene inactivation by disruption, removal, modification, orreplacement of target gene sequences. Methods for gene inactivation arewell known in the art. For example, a target gene can be inactivated bythe insertion of a heterologous sequence (such as a selectable markerand/or a stop codon) into a target gene, deletion of a part of a gene orthe entire gene, modification of a gene (e.g., by frame shift mutation,nonsense mutation, missense mutation, point mutation, replacement of apart or a whole gene with another nucleic acid sequence), or acombination of any of the above.

Inserted sequences can replace previously existing sequences in a geneor can be added to such sequences, depending on the design of thetargeting construct. The design of targeting constructs can be altered,depending upon whether it is desired to completely knock out thefunction of a gene or to maintain some reduced level of function. In thecase of SIGLEC1, complete knock out of function is desirable. By way ofexample and not of limitation, the SIGLEC1 gene can be knocked out bydeletion of part of exon 1 and all of exons 2 and 3, for example byreplacing part of exon 1 and all of exons 2 and 3 with a neomycinselectable cassette. In some embodiments, the modification can alsoinclude the addition of LoxP sites on either side of a sequence of aSIGLEC1 gene that is to be mutated. In some instances, the targetingconstruct can contain both the loxP sites flanking a sequence to beinserted and the CRE recombinase. In other cases, a two-vector systemcan be used, wherein the targeting construct includes the loxP sitesflanking a sequence to be inserted and a second vector includes atransgene encoding for the CRE recombinase. The transgene for CRErecombinase can be placed under the influence of a tissue-specificpromoter, such that its expression renders the SIGLEC1 genenon-functional in specific cell lineages. Similarly, the antibioticselectable cassette can be flanked by loxP sites so that it can bedeleted at a later stage using the Cre recombinase.

In the case of CD163, it is desirable to only inactivate its functionrelated to PRRSV binding and/or uncoating while leaving the otherfunctions of CD163 minimally affected or unaffected. While not beingbound to any particular theory, it is believed that a complete CD163knock-out may not be viable or could be seriously compromised due to therole of CD163 in binding and internalization of haemoglobin-haptoglobincomplexes. Accordingly, CD163 can be inactivated by disrupting the fifthN-terminal scavenger receptor cysteine-rich (SRCR) domain of CD163,which was previously shown to play a leading role in PRRSV infection(Van Gorp et al., 2010), while leaving the other domains unaffected.SRCR domain 5 can be genetically modified by, e.g., introducing pointmutations which alter the structure of this domain or by swapping thisdomain with another one. For example, SRCR domain 5 can be replaced withSRCR domain 8 from CD163 Ligand (CD163L) since this domain “swap” hasbeen shown to reduce relative PRRSV infectivity to 0% in cultured cells(Van Gorp et al 2010).

In other approaches, the coding sequences for a target gene are notaltered or are minimally altered and, rather, sequences impactingexpression of a target gene, such as promoter sequences, are targeted.In any case, selectable marker insertion is often desirable tofacilitate identification of cells in which targeting has occurred. Ifdesired, such markers or other inserted sequences can later be removedby, e.g., Cre-Lox or similar systems.

Targeted gene modification uses nucleic acid constructs which haveregions of homology with a target gene (e.g., SIGLEC1 or CD163) or withthe flanking regions of a target gene, such that integration of theconstruct into the genome alters expression of the gene, either bychanging the sequence of the gene and/or by altering the levels ofexpression of the gene. Thus, to alter a gene, a targeting construct isgenerally designed to contain three main regions: (i) a first regionthat is homologous to the locus to be targeted (e.g., the SIGLEC1 orCD163 gene, or a flanking sequence thereof), (ii) a second region thatis a heterologous polynucleotide sequence (e.g., encoding a selectablemarker, such as an antibiotic resistance protein) that is tospecifically replace a portion of the targeted locus or is inserted intothe targeted locus, and (iii) a third region that, like the firstregion, is homologous to the targeted locus, but typically is notcontiguous with the first region of the construct. Homologousrecombination between the targeting construct and the targeted wild-typelocus results in deletion of any locus sequences between the two regionsof homology represented in the targeting vector and replacement of thatsequence with, or insertion into that sequence of, a heterologoussequence (e.g., a heterologous sequence that encodes a selectablemarker). An exemplary construct and vector for carrying out suchtargeted modification is described in Example 1; however, other vectorsthat can be used in such approaches are known in the art and can readilybe adapted for use in the invention.

In order to facilitate homologous recombination, the first and thirdregions of the targeting vectors (see above) include sequences thatexhibit substantial sequence identity to the genes to be targeted (orflanking regions). For example, the first and third regions of thetargeting vectors can have sequences that are at least about 80%, atleast about 90%, at least about 95%, at least about 98%, at least about99%, or about 100% identical to the targeted genes or flanking regions.Sequence identity is typically measured using BLAST® (Basic LocalAlignment Search Tool) or BLAST® 2 with the default parameters specifiedtherein (see, Altschul et al., J. Mol. Biol. 215:403-410, 1990; Tatianaet al., FEMS Microbiol. Lett. 174:247-250, 1999). Thus, sequences havingat least about 80%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or even about 100% sequence identity withthe targeted gene loci can be used in the invention to facilitatehomologous recombination.

The total size of the two regions of homology (i.e., the first and thirdregions described above) can be, for example, about 2 kilobases (kb) toabout 25 kb (e.g., about 4 kb to about 20 kb, about 5 kb to about 15 kb,or about 6 kb to about 10 kb). The size of the region that replaces aportion of the targeted locus or is inserted into the targeted locus(the second region as described above) can be, for example, about 0.5 kbto about 5 kb (e.g., about 1 kb to about 4 kb or about 3 to about 4 kb).

Various types of targeting construct delivery methods can be used. Celltransfection methods, including calcium phosphate, lipofection,electroporation, and nuclear injection can be used to deliver thetargeting construct. If the gene is transcriptionally active in the celltype being used, then a promoterless selectable marker strategy can beemployed, so that antibiotic resistance will only be found in cells thathave had a recombination event within a transcribed unit. Alternatively,if the gene is transcriptionally inactive in the cell type being used,promoters that are inducible, tissue-specific or contain insulators suchas matrix attachment regions (MARs) can be used.

Alternatively, siRNA technology can be used to “silence” the transcriptfor SIGLEC1. Antisense technology is well known in the art. Briefly, anucleotide sequence generally containing between about 19 and about 29nucleotides which is complementary to the sense mRNA sequence of SIGLEC1is used. The degree of complementarity generally ranges from about 70%to about 100%. Preferably, complementarity is greater than about 80%,more preferably greater than about 90%, and even more preferably greaterthan about 95%. Regions of SIGLEC1 mRNA that are suitable for targetingwith siRNA can be readily determined by comparing the efficacy ofseveral antisense sequences designed to be complementary to differentregions of SIGLEC1 mRNA in preventing production of the SIGLEC1 protein.Such experiments can be readily performed without undue experimentationby any of the known techniques in the art.

Vectors used for siRNA expression are well known in the art. The vectormay be any circular or linear length of DNA that either integrates intothe host genome or is maintained in episomal form. In general, siRNAexpression cassettes may be ligated into a DNA transfer vector, such asa plasmid, or a lentiviral, adenoviral, alphaviral, retroviral or otherviral vector. Exemplary mammalian viral vector systems includeadenoviral vectors; adeno-associated type 1 (“AAV-1”) oradeno-associated type 2 (“AAV-2”) viral vectors; hepatitis deltavectors; live, attenuated delta viruses; herpes viral vectors;alphaviral vectors; or retroviral vectors (including lentiviralvectors).

Transformation of mammalian cells can be performed according to standardtechniques known in the art. Any of the well-known procedures forintroducing foreign nucleotide sequences into host cells can be used solong as they successfully introduce at least the siRNA construct intothe host cell. These procedures include the use of viral transduction(for example, by use of any of the viral vectors listed in the precedingparagraph, e.g., by retroviral infection, optionally in the presence ofpolybrene to enhance infection efficiency), calcium phosphatetransfection, electroporation, biolistic particle delivery system (i.e.,gene guns), liposomes, microinjection, and any of the other knownmethods for introducing cloned genomic DNA, cDNA, synthetic DNA or otherforeign genetic material into a host cell. In the present invention, thesiRNA directed against SIGLEC1 is introduced into a donor swinefibroblast cell, which is subsequently used to produce a transgenicallymodified swine of the present invention.

The transgenic animals of the invention can be made using the followinggeneral somatic cell nuclear transfer procedure. Briefly, the genome ofa somatic porcine cell (e.g., a fetal fibroblast) is geneticallymodified by gene targeting as described above, to create a donor cell.The nucleus of such a genetically modified donor cell (or the entiredonor cell, including the nucleus) is then transferred into a recipientcell, for example, an enucleated oocyte. The donor cell can be fusedwith a enucleated oocyte, or donor nucleus or the donor cell itself canbe injected into the recipient cell or injected into the perivitellinespace, adjacent to the oocyte membrane.

Thus, upon obtaining somatic cells in which a target gene has beentargeted (one or both alleles, as described above), nuclear transfer canbe carried out. Optionally, the genetically modified donor cells can becryopreserved prior to nuclear transfer. Recipient cells that can beused include oocytes, fertilized zygotes, or the cells of two-cellembryos, all of which may or may not have been enucleated.

Recipient oocytes can be obtained using methods that are known in theart or can be purchased from commercial sources (e.g., BoMed Inc.,Madison, Wis.). The oocyte can be obtained from a “gilt,” a female pigthat has never had offspring or from a “sow,” a female pig that haspreviously produced offspring.

Accordingly, a genetically modified swine wherein at least one allele ofa SIGLEC1 gene has been inactivated can be produced by enucleating aswine oocyte; fusing the oocyte with a donor swine fibroblast cell, thegenome of the fibroblast cell comprising at least one inactivatedSIGLEC1 allele; and activating the oocyte to produce an embryo.Similarly, a genetically modified swine wherein at least one allele of aCD163 gene has been inactivated, wherein inactivation of the CD163allele results in a CD163 protein which cannot bind and/or uncoat aporcine reproductive and respiratory syndrome virus (PRRSV) can beproduced by enucleating a swine oocyte; fusing the oocyte with a donorswine fibroblast cell, the genome of the fibroblast cell comprising atleast one inactivated CD163 allele; and activating the oocyte to producean embryo. Both methods can further include a step of transferring theembryo into a reproductive tract of a surrogate swine, wherein thesurrogate swine has initiated estrus but has not yet completedovulation; and wherein gestation and term delivery of the embryoproduces a genetically modified swine whose genome comprises at leastone inactivated SIGLEC1 and/or CD163 allele. The present invention alsorelates to progeny of such genetically modified swine wherein at leastone allele of a SIGLEC1 gene has been inactivated and/or wherein atleast one allele of a CD163 gene has been inactivated, whereininactivation of the CD163 allele results in a CD163 protein which cannotbind and/or uncoat a porcine reproductive and respiratory syndrome virus(PRRSV).

Methods for enucleating swine oocytes are known in the art, andenucleation can be achieved by any of the standard methods. For example,enucleating a oocyte can be achieved with a micropipette in amicromanipulation medium.

Introduction of a membrane-bound nucleus from a donor swine cell into anenucleated recipient swine oocyte to form an oocyte containing the donornucleus can be performed by fusing together the membrane of themembrane-bound nucleus from the donor mammalian cell with the membraneof the enucleated recipient mammalian oocyte to form an oocytecontaining the nucleus from the donor mammalian cell. Alternatively,such introduction can be performed by microinjecting themembrane-bounded nucleus from the mammalian donor cell into theenucleated recipient mammalian oocyte to form an oocyte containing thenucleus from the donor mammalian cell. For example, one can introduce adonor cell (or nucleus) into the space under the zona pellucida or intothe perivitelline space of the enucleated, recipient oocyte, andsubsequently carry out membrane fusion to produce an oocyte containingwithin its cytoplasm the donor nucleus. All means of introducing donornuclear material into an enucleated recipient mammalian oocyte known tothose of ordinary skill in the art are useful in the methods disclosedherein.

For example, the fusing step can be performed in a fusion medium.Alternatively, an inactivated virus or a fuseogenic agent such aspolyethylene glycol (PEG) can be used to promote fusion. See, e.g.,Graham (1969) Wistar Inst. Symp. Monogr. 9:19-33, and McGrath et al.(1983) Science 220:1300-1302 for the use of viruses; Fisher et al.(1981) Tech. Cell. Physiol. 1:1-36 for chemically induced cell fusion;and Berg (1982) Bioelectrochem. Bioenerg. 9:223-228, and Robl et al.(1987) J. Anim. Sci. 64:642-647 for electrically induced cell fusion.

U.S. Pat. No. 6,211,429 B1, the contents of which are herebyincorporated by reference, describes methods for in vitro and in vivodevelopment of activated oocytes. The term “activated” or “activation”refers to the capacity of an unfertilized oocyte to develop to at leastthe pronuclear stage, or beyond, after treatment with anoocyte-modifying agent and a reducing agent. Generally speaking, thepronuclear stage is achieved about three to seven hours after suchtreatment. The term “oocyte-modifying agent” refers to an agent that canreact with a substrate on or in an oocyte, for example a thiol (—SH)group, which can be a protein thiol group; the effect of this reaction,when followed by treatment of the oocyte with a reducing agent accordingto the methods disclosed in U.S. Pat. No. 6,211,429 B1, results inactivation of mammalian oocytes.

The combined use of an —SH or oocyte-modifying agent such as thimerosaland an —SH reducing agent such as dithiothreitol is able to inducecomplete activation of mammalian oocytes. Coupling of a short treatmentwith thimerosal with only a single calcium transient before treatmentwith a reducing agent such as DTT is particularly effective in achievingactivation. Thimerosal triggers a series of Ca²⁺ spikes in mammalianoocytes, which when followed by an incubation with a reducing agent suchas DTT can stimulate pronuclear formation. Thus, after thimerosal iswashed out, DTT is then added to reverse the actions of thimerosal,followed by washing it out to allow the embryo to continue development.The combined thimerosal/DTT treatment also induces cortical granuleexocytosis, subsequent hardening of the zona pellucida, and developmentof the activated oocytes to the blastocyst stage.

In addition to thimerosal, other oocyte-modifying agents can be used,such as t-butyl hydroperoxide; thiourea; phenylephrine;N-aklylmaleimides such as N-ethylmaleimide; oxidized glutathione;alpha-haloacids such as iodoacetate, chloroacetate, and bromoacetate;iodoacetamide; p-mercuribenzoate; p-chloromercuribenzoate;5,5′-dithiobis(2-nitrobenzoic acid) (DTNB);(2-trimethylammonium)ethylmethanethiosulfonate (MTSET); and(2-sulfonatoethyl)methanethiosulfonate (MTSES). Useful reducing agents,such as thiol (—SH) group reducing agents, in addition to DTT, includebut are not limited to dithioerythritol (DTE); beta-mercaptoethanol;cysteine; reduced glutathione; reduced thiourea; thioglycolate; andascorbic acid.

The time period during which oocytes are contacted with theoocyte-modifying agent is a period effective to result in activation ofthe oocytes when followed by treatment with a reducing agent. Such timeperiod can be in the range of from about 5 minutes to about 20 minutes,preferably from about 5 minutes to about 15 minutes, or more preferablyfrom about 5 minutes to about 12 minutes. The time period during whichthe oocytes are contacted with the reducing agent is suitably longenough to result in activation of the oocytes when preceded by treatmentwith an oocyte-modifying agent. Such time period can be in the range offrom about 5 minutes to about 1 hour, preferably from about 10 minutesto about 45 minutes, more preferably from about 20 minutes to about 40minutes, and still more preferably about 30 minutes.

Contacting of the enucleated oocyte with the reducing agent followingthe contacting with the oocyte-modifying agent can occur substantiallyimmediately, or can occur within a time interval in the range of fromabout 5 seconds to about 5 minutes after exposure of the oocyte to theoocyte-modifying agent. The oocyte-modifying agent-treated oocyte can betransferred into medium containing the reducing agent without anyintermediate wash step. Alternatively, the oocyte-modifyingagent-treated oocyte can be washed in control or reducingagent-containing medium to substantially remove oocyte-modifying agentbefore culturing the oocyte in reducing agent-containing medium. Asanother alternative, the reducing agent can be added directly to theoocyte while the latter is still present in oocyte-modifyingagent-containing medium.

Alternatively, oocyte activation can also be achieved with a number ofother chemical treatments that do not involve calcium, e.g., proteinkinase inhibition (Mayes et al. (1995) Biol. Reprod. 53:270-275), orinhibition of protein synthesis (Nussbaum et al. (1995) Mol. Reprod.Dev. 41:70-75).

After activation, the oocyte is typically cultured for a brief period invitro. The resulting embryo is then transferred into a surrogate female,and development of the embryo proceeds in the surrogate. For example,the embryos can be cultured for about a week, and then transferredsurgically or non-surgically to the reproductive tract of a surrogate.The embryos can be transferred into an oviduct through an ovarianfimbria of the surrogate. Alternatively, the embryos can be transferredinto an oviduct of a surrogate by using a catheter that penetrates thewall of the oviduct. Another way of transferring embryos involvesculturing them until the blastocyst stage followed by introduction intothe reproductive tract of a surrogate swine. These methods are wellknown in the art, and can readily be applied in producing thegenetically modified swine of the present invention.

Additional methods for making genetically modified swine and other largeanimals are known in the art and can also be used in the presentinvention (see, e.g., US 2005/0120400 A1; U.S. Pat. No. 5,945,577; WO96/07732; WO 97/07669; WO 97/07668; WO 2005/104835; Lai et al.,Reproductive Biology and Endocrinology 1:82, 2003; Hao et al.,Transgenic Res. 15:739-750, 2006; Li et al., Biology of Reproduction75:226-230, 2006; Lai et al., Nature Biotechnology 24 (4):435-436, 2006;Lai et al., Methods in Molecular Biology 254 (2):149-163, 2004; Lai etal., Cloning and Stem Cells 5 (4):233-241, 2003; Park et al., AnimalBiotechnology 12 (2):173-181, 2001; Lai et al., Science 295:1089-1092,2002; Park et al., Biology of Reproduction 65:1681-1685, 2001; thecontents of each of which are incorporated herein by reference).

Other feasible methods for producing genetically modified swine includeinjection or transduction of nucleases (zinc-finger nucleases, or Talnucleases that would target the gene of interest) into somatic cells,followed by somatic cell nuclear transfer and transfer of the embryointo a surrogate. In addition, sperm- or intracytoplasmic sperminjection (ICSI)-mediated genetic modification can also be employed.Briefly, in ICSI-mediated modification, a targeting construct is mixedwith the sperm, and both are injected into an oocyte. In sperm-mediatedmodification, the construct is mixed with the sperm, and in vitrofertilization (IVF) or insemination is used to impregnate a surrogate. Askilled artisan can readily adjust these methods to production ofknockout pigs of the present invention. While not fully developed forthe pig, embryonic stem cell technology, or induced pluripotent cells,as have been developed for mouse, may be genetically modified and usedeither as donor cells for somatic cell nuclear transfer or for theproduction of chimeric animals.

As mentioned above, the present invention is also directed togenetically modified swine in which (1) both alleles of SIGLEC1 genehave been inactivated, (2) both alleles of CD163 genes have beeninactivated, wherein inactivation of the CD163 alleles results in aCD163 protein which cannot bind and/or uncoat a porcine reproductive andrespiratory syndrome virus (PRRSV), or (3) both alleles of SIGLEC1 andboth alleles of CD163 have been inactivated. Genetically modified swinewhich are homozygous for gene inactivation can be produced by matingswine heterozygous for gene inactivation, and screening the progeny toidentify animals which are homozygous for inactivation of the gene(s).The present invention is also directed to progeny of such geneticallymodified swine, wherein one or both alleles of a SIGLEC1 gene have beeninactivated and/or wherein one or both alleles of a CD163 gene have beeninactivated, wherein inactivation of the CD163 alleles results in aCD163 protein which cannot bind and/or uncoat a porcine reproductive andrespiratory syndrome virus (PRRSV).

Accordingly, the present invention is directed to a method for producinga genetically modified swine wherein both alleles of a SIGLEC1 gene havebeen inactivated by mating a female genetically modified swine whereinat least one allele of a SIGLEC1 has been inactivated with a malegenetically modified swine wherein at least one allele of SIGLEC1 hasbeen inactivated to produce F1 progeny; and screening the F1 progeny toidentify genetically modified swine wherein both alleles of the SIGLEC1gene have been inactivated. Similarly, the present invention is directedto a method for producing a genetically modified swine wherein bothalleles of a CD163 gene have been inactivated by mating a femalegenetically modified swine wherein at least one allele of CD163 has beeninactivated with a male genetically modified swine wherein at least oneallele of CD163 has been inactivated to produce F1 progeny; andscreening the F1 progeny to identify genetically modified swine whereinboth alleles of the CD163 gene have been inactivated.

The present invention also provides methods for producing a geneticallymodified swine wherein both alleles of a SIGLEC1 gene and both allelesof a CD163 gene have been inactivated. One such method comprises matinga genetically modified swine wherein at least one allele of a SIGLEC1gene has been inactivated with a genetically modified swine wherein atleast one allele of a CD163 gene has been inactivated to produce F1progeny; screening the F1 progeny to identify genetically modified swinewherein at least one allele of the SIGLEC1 gene has been inactivated andat least one allele of the CD163 gene has been inactivated; mating thegenetically modified swine wherein at least one allele of the SIGLEC1gene has been inactivated and at least one allele of the CD163 gene hasbeen inactivated to each other produce F2 progeny; and screening the F2progeny to identify genetically modified swine wherein both alleles ofthe SIGLEC1 gene and both alleles of the CD163 gene have beeninactivated.

Methods for producing genetically modified swine wherein at least oneallele of a SIGLEC1 and/or at least one allele of a CD163 gene have beeninactivated are disclosed in the foregoing sections. Screening ofprogeny can be performed as is standard in the art, e.g., by using PCRor Southern blotting.

Another method for producing a genetically modified swine wherein bothalleles of a SIGLEC1 gene and both alleles of a CD163 gene have beeninactivated comprises mating a genetically modified swine homozygous forSIGLEC1 inactivation with a genetically modified swine homozygous forCD163 inactivation to produce F1 progeny, mating the F1 progeny togenerate F2 progeny, and screening the F2 progeny to identify animalshomozygous for both SIGLEC1 and CD163 inactivation.

Yet another method for producing a genetically modified swine whereinboth alleles of a SIGLEC1 gene and both alleles of a CD163 gene havebeen inactivated comprises mating a genetically modified swine whereinat least one allele of a SIGLEC1 gene and at least one allele of a CD163gene have been inactivated to another genetically modified swine whereinat least one allele of a SIGLEC1 gene and at least one allele of a CD163gene have been inactivated to produce F1 progeny; and screening the F1progeny to identify genetically modified swine wherein both alleles ofthe SIGLEC1 gene and both alleles of the CD163 gene have beeninactivated.

The present invention also relates to progeny of genetically modifiedswine produced by any of the methods above, wherein one or both allelesof a SIGLEC1 gene have been inactivated and/or wherein one or bothalleles of a CD163 gene have been inactivated, wherein inactivation ofthe CD163 gene results in a CD163 protein which cannot bind and/oruncoat a porcine reproductive and respiratory syndrome virus (PRRSV).

In addition to being obtainable by breeding approaches involvingheterozygous animals, homozygous mutant animals can also be obtainedusing an approach in which a cell (e.g., a fetal fibroblast) including amutation in one allele, such as a cell obtained from an animal producedusing the method summarized above, is subjected to gene targeting byhomologous recombination to achieve modification of the remainingallele. The resulting donor cell can then be used as a source of amodified nucleus for nuclear transfer into a recipient cell, such as anenucleated oocyte, leading to the formation of a homozygous mutantembryo which, when implanted into a surrogate female, develops into ahomozygous mutant animal. Genetically modified swine wherein bothalleles of SIGLEC1 and/or cD163 gene(s) have been inactivated can alsobe produced by injecting or transducing zinc finger nucleases or Talnucleases (which can target both alleles of a gene at the same time)into somatic cells, followed by somatic cell nuclear transfer (SCNT) andembryo transfer into a surrogate to produce such swine. The presentinvention also relates to progeny of such genetically modified swine,wherein one or both alleles of a SIGLEC1 gene have been inactivatedand/or wherein one or both alleles of a CD163 gene have beeninactivated, wherein inactivation of the CD163 gene results in a CD163protein which cannot bind and/or uncoat a porcine reproductive andrespiratory syndrome virus (PRRSV).

Having described the invention in detail, it will be apparent thatmodifications and variations are possible without departing from thescope of the invention defined in the appended claims.

EXAMPLES

The following non-limiting examples are provided to further illustratethe present invention.

Example 1

Modification of the SIGLEC1 gene

The approach employed to ablate the sialoadhesin gene made use ofhomologous recombination to remove protein coding exons and introducepremature stops in the remaining coding sequence of the sialoadhesingene. The porcine sialoadhesin gene (SIGLEC1, NCBI reference sequenceNM_214346) encodes a 210-kDa protein from an mRNA transcript of 5,193bases (Vanderheijden et al. 2003). Porcine genomic sequence from theregion around the sialoadhesin gene (Genbank accession no. CU467609) wasused to generate oligonucleotides to amplify genomic fragments byhigh-fidelity PCR [AccuTaq (Invitrogen)] for generation of a targetingconstruct. One fragment (the ‘upper arm’) included the first coding exonand the 3304 bp upstream from the translation start. The second (‘lowerarm’) fragment was 4753 bp in length and included most of the introndownstream of the third coding exon and extended into the 6th intron(including the 4th, 5th and 6th coding exons). Based on comparisons withthe mouse and human sialoadhesin genomic sequences, the porcinesialoadhesin gene was predicted to be composed of 21 exons (panels A andB of FIG. 1). Exon 2 is conserved between pig, mouse, and human. Anamino acid alignment of exon 2 revealed that the six amino acids inmouse sialoadhesin known to be associated with sialic acid bindingactivity were conserved in pig sialoadhesin (panel C of FIG. 1). Theinitial targeting strategy focused on creating alterations in thesialoadhesion gene such that no functional protein was expected to beobtained from the mutated gene. Other inactivation strategies couldinclude targeted modification of selected residues in exon 2 of thesialoadehesin gene or altering the immunoglobulin domains in wayspredicted to preclude PRRS virus binding (possibly by changing theirorder or by substituting them for comparable domains from otherspecies). Additional modifications may include flanking of the neomycincassette or one of the immunoglobulin-like domains with loxP sites topermit inducible or tissue-specific removal if desirable.

In the current gene disruption, part of exon 1 and all of exons 2 and 3were replaced with a neomycin selectable cassette by using areplacement-type vector (panel D of FIG. 1) (Mansour et al. 1988). Inthe plasmid construct, the phosphoglycerol kinase (PGK) promoter wasused to drive expression of the neomycin cassette to permit positiveselection of transfected colonies.

Donor Cell Preparation

A male fetal fibroblast primary cell line, from day 35 of gestation, wasisolated from large commercial white pigs (Landrace). The cells werecultured and grown for 48 hours to 80% confluence in Dulbeco's ModifiedEagles Medium (DMEM) containing 5 mM glutamine, sodium bicarbonate (3.7g/L), penicillin-streptomycin and 1 g/L d-glucose, and supplemented with15% Hyclone's Fetal bovine Serum, 10 μg/ml gentamicin, and 2.5 ng/mlbasic fibroblast growth factor (Sigma). The media was removed andreplaced with fresh media four hours prior to the transfection.Fibroblast cells were washed with 10 mls of phosphate buffered saline(DPBS; Invitrogen) and lifted off the 75 cm² flask with 1 ml of 0.05%trypsin-EDTA (Invitrogen). The cells were resuspended in DMEM andcollected by centrifugation at 600×g for 10 mins. The cells were washedwith Opti-MEM (Invitrogen) and centrifuged again at 600×g for 10minutes. Cytosalts (75% cytosalts [120 mM KCl, 0.15 mM CaCl₂, 10 mMK₂HPO₄; pH 7.6, 5 mM MgCl₂]) and 25% Opti-Mem were used to resuspend thepellet (van den Hoff et al. 1992). The cells were counted with ahemocytometer and adjusted to 1×10⁶ cells per ml. Electroporation of thecells was performed with 4 μg of single stranded targeting DNA (achievedby heat denaturation) in 200 μl of transfection media consisting of1×10⁶ cells/ml. The cells were electroporated in a BTX ECM 2001 electrocell manipulator by using three 1 msec pulses of 250V. Theelectroporated cells were diluted in DMEM/FBS/FGF at 10,000 cells per 13cm plate. The electroporated cells were cultured overnight withoutselective pressure. The following day, media was replaced with culturemedia containing G418 (0.6 mg/ml). After 10 days of selection,G418-resistant colonies were isolated and transferred to 24-well platesfor expansion. After growth in the 24-well plates, the cells were split(˜half were used for genomic DNA isolation) into 6-well plates. PCR wasused to determine if targeting of the sialoadhesin gene was successful.The reaction used oligonucleotides that annealed in thephosphoglucokinase (PGK) cassette (which includes Neo) paired with theones that annealed to genomic DNA in the sialoadhesin locus that wasbeyond the region of the targeting arms (see FIG. 2). Successfultargeting of both ‘arms’ was assessed in this way. A targeted fibroblastclone (4-18) was identified and some of the cells in the culture wereused for nuclear transfer (see below) while others were frozen forfuture use. Southern blot verification of homologous recombination wasused to provide additional confirmation of successful targeting.

Collection of Oocytes and In Vitro Maturation (IVM)

Pig oocytes were purchased from ART Inc (Madison, Wis.) and maturedaccording to the supplier's instructions. After 42-44 h of in vitromaturation, the oocytes were stripped of their cumulus cells by gentlevortexing in 0.5 mg/ml hyaluronidase. After removal of the cumuluscells, oocytes with good morphology and a visible polar body (metaphaseII) were selected and kept in the micromanipulation medium at 38.5° C.until nuclear transfer.

Somatic Cell Nuclear Transfer, Fusion/Activation of Nuclear TransferComplex Oocytes, and In Vitro Developmental Culture

Under a microscope, a cumulus-free oocyte was held with a holdingmicropipette in drops of micromanipulation medium supplemented with 7.5μg/mL cytochalasin B and covered with mineral oil. The zona pellucidawas penetrated with a fine glass injecting micropipette near the firstpolar body, and the first polar body and adjacent cytoplasm, presumablycontaining the metaphase-II chromosomes, was aspirated into the pipette,the pipette was withdrawn and the contents discarded. A single, round,and bright donor cell with a smooth surface was selected and transferredinto the perivitelline space adjacent to the oocyte membrane (Lai et al.2006; Lai et al. 2002).

The nuclear transfer complex (oocyte+fibroblast) was fused in fusionmedium with a low calcium concentration (0.3 M mannitol, 0.1 mMCaCl₂.2H₂O, 0.1 mM MgCl₂.6H₂O and 0.5 mM HEPES). The fused oocytes werethen activated by treatment with 200 μM thimerosal for 10 minutes in thedark, followed by rinsing and treatment with 8 mM dithiothreitol (DTT)for 30 minutes; the oocytes were then rinsed again to remove the DTT(Macháty and Prather 2001; Machaty et al. 1997). Followingfusion/activation, the oocytes were washed three times with porcinezygote medium 3 (PZM3) supplemented with 4 mg/mL of BSA (Im et al.2004), and cultured at 38.5° C. in a humidified atmosphere of 5% O₂, 90%N₂ and 5% CO₂ for 30 min. Those complexes that had successfully fusedwere cultured for 15-21 hours until surgical embryo transfer to asurrogate.

Surrogate Preparation, Embryo Transfer, Pregnancy Diagnosis and Delivery

The surrogate gilts were synchronized by administering 18-20 mgREGU-MATE (altrenogest 0.22% solution) (Intervet, Millsboro, Del.) mixedinto the feed for 14 days using a scheme dependent on the stage of theestrous cycle. After the last REGU-MATE treatment (105 hours), an IMinjection of 1000 units of hCG was given to induce estrus. Othersurrogates that had naturally cycled on the appropriate date were alsoincluded in the surrogate pool. Surrogates on the day of (day 0), or thefirst day after, standing estrus were used (Lai et al. 2002). Thesurrogates were aseptically prepared and a caudal ventral incision wasmade to expose the reproductive tract. Embryos were transferred into oneoviduct through the ovarian fimbria. Surrogates were checked forpregnancy by abdominal ultrasound examination around day 30, and thenchecked once a week through gestation. Parturition is in the pig isgenerally at 114 days of gestation.

After transfection and screening of fetal fibroblast cells candidatedonor cells were identified and used for somatic cell nuclear transfer(SCNT). Six hundred and sixty-six SCNT embryos were transferred to twosurrogates. One delivered 6 normal male piglets on day 115 of gestation,and a C-section was performed on the other surrogate on day 117 ofgestation resulting in two normal male piglets, as show in Table 1.

TABLE 1 Embryo transfer results from using SIGLEC1 +/− male donor cells.# Surro- Trans- Date gate Genotype ferred Results Sep. 23, 2010 O963SIGLEC1^(+/−) 388 6 normal male piglets ♂ born Jan. 16, 2011 Sep. 24,2010 O962 SIGLEC1^(+/−) 278 2 normal male piglets ♂ delivered byC-section Jan. 18, 2011

FIG. 2 shows the organization of the sialoadhesin (SIGLEC1) gene,targeting vector and expected recombined genotype. The top panel of FIG.2 shows the targeting construct used for homologous recombination. Asdescribed above, the ‘upper’ DNA fragment used to create the targetingconstruct contained ˜3.5 kb upstream of exon 1 and included part of exon1 (after the start codon). The ‘lower’ DNA fragment began within intron3 and included exons 4, 5, 6 and part of exon 7. Most of exon 1 and allof exons 2 & 3 were substituted with a neomycin (neo) cassette; athymidine kinase (TK) cassette was available immediately downstream ofthe lower arm for use as a negative selectable marker if necessary. Thebottom panel of FIG. 2 illustrates the mutated sialoadhesin genefollowing homologous recombination. The arrows represent oligonucleotidebinding sites used for PCR-based screening of targeted cell lines andcloned pigs. Targeting by the upper and lower arms was performed byoligonucleotides annealing as shown by arrows representing primers“upper sialo targeting C” and “PGK polyA reverse” (SEQ ID NOs: 1 and 3)and “PGK promoter forward” and “7Rw1” (SEQ ID NOs: 4 and 6),respectively. The oligonucleotide sequences are listed in Table 2 below.An additional check PCR made use of oligonucleotides that flanked theablated region/Neo cassette (primers “Exon 1 end ck” and “Intron 3 ckReverse” (SEQ ID NOs: 2 and 5, respectively)). There is approximately a500 bp difference in product size between the disrupted allele and thewild-type allele.

TABLE 2 Primer names and sequences used in the project.upper sialo targeting C ggaacaggctgagccaataa (SEQ ID NO: 1)Exon 1 end ck gcattcctaggcacagc (SEQ ID NO: 2) PGK polyA reverseggttctaagtactgtggtttcc (SEQ ID NO: 3) PGK promoter forwardagaggccacttgtgtagcgc (SEQ ID NO: 4) Intron 3 ck Reversectccttgccatgtccag (SEQ ID NO. 5) 7Rw1 caggtaccaggaaaaacgggt(SEQ ID NO: 6)The primers in Table 2 were assigned sequence ID numbers based on theposition of the arrows in the bottom panel of FIG. 2 going from left toright. Thus, the left-most arrow in the bottom panel of FIG. 2 shows thelocation of the “upper sialo targeting C” primer (SEQ ID NO: 1), thenext arrow to the right shows the location of the “Exon 1 end ck” primer(SEQ ID NO: 2), the next arrow to the right of that shows the locationof the “PGK polyA reverse” primer (SEQ ID NO: 3), and so forth.

Screening for SIGLEC1 Inactivation

Genomic DNA was isolated from the piglets and used to confirm thetargeting events. Successful targeting of the SIGLEC1 gene wasdetermined by using PCR with oligonucleotides that annealed within theNeo cassette paired with oligonucleotides that annealed to SIGLEC1genomic DNA that was beyond the region contained within the targetingconstruct. In the top panel of FIG. 3, targeting by the ‘upper’ arm wasexamined by using the ‘PGK polyA reverse’ and ‘upper sialo targeting C’oligonucleotides (SEQ ID NOs. 3 and 1, respectively; illustrated in FIG.2). A product of the expected size (˜4500 bp) was produced. In thebottom panel, successful targeting by the ‘lower’ arm was determined byusing the ‘PGK promoter forward’ and ‘7Rw1’ oligonucleotides (SEQ IDNOs: 4 and 6, respectively; illustrated in FIG. 2). A product of theexpected size (˜5000 bp) was produced. The ‘lower arm plasmid’ controlwas a partial construct containing the Neo cassette with a sialoadhesingene fragment representing most of intron 3 and most of exon 7. The 7Rw1oligonucleotide was able to anneal to the exon 7 sequence present in theplasmid, and along with the PGK promoter forward oligonucleotide, it wasable to produce a product that was identical to what would be producedfrom a successful targeting event. Both panels illustrate targeting PCRreactions performed on genomic DNA extracted from eight piglet clonesgenerated from the 4-18 targeted fetal fibroblast line.

Detection of both wild-type and targeted sialoadhesin alleles wasperformed by using PCR with oligonucleotides that annealed to DNAflanking the targeted region of the sialoadhesin gene. The ‘Exon 1 endck’ and ‘Intron 3 ck Reverse’ oligonucleotides were used (SEQ ID NOs: 2and 5, respectively; illustrated in FIG. 2). The products generated were2400 bp for the wildtype allele and ˜2900 bp for the targeted allele. InFIG. 4, the panel on the left (panel A of FIG. 4) illustrates testreactions performed on wild-type genomic DNA, the targeting plasmid usedfor transfections, genomic DNA (4-18) from a successfully targetedfibroblast clone (note the two bands) and genomic DNA (4-3) from anon-targeted fibroblast clone. The panel on the right (panel B of FIG.4) illustrates the reaction performed on genomic DNA extracted fromeight piglet clones generated from the 4-18 targeted fetal fibroblastline. There were two PCR products of the expected sizes in each lane,while the wildtype DNA and targeting plasmid templates only had a singleband. Thus, all of the piglets produced by SCNT were heterozygous forthe intended mutation, i.e. SIGLEC+/−.

Production of Homozygous Animals

Male genetically modified swine, which were identified as heterozygousfor SIGLEC1 inactivation were used as male founders (F0 generation), andwere mated to wild-type females to produce male and female animals thathad one inactivated SIGLEC1 allele (F1). The F1 males were then mated tothe F1 females to produce F2 progeny, which have both SIGLEC1 allelesinactivated. Such animals can be identified following birth by the PCRdescribed above for SIGLEC1 or alternatively by Southern blotting.

Example 2

Generation of a CD163 Targeting Construct

As already established, deletion of the cytoplasmic domain of CD163eliminates infectivity of PRRSV, as does the deletion or modification ofSRCR domain 5. Since some of the SRCR domains of CD163 have importantfunctions for survival of the animal, e.g. hemoglobin removal,modification of the gene such that these other functions remain intactrepresents a solid strategy to create pigs that are resistant to PRRSV.Prior research has also suggested that the replacement of SRCR5 domainwith CD163L domain 8 also blocks infectivity (Van Gorp et al. 2010b).Thus, a targeting construct can be designed as shown in FIG. 6 to swapout SRCR5 of CD163 with the SRCR domain CD163L.

Once the targeting construct is made, the genetically modified swinewhich are heterozygous for inactivated CD163, wherein inactivation ofthe CD163 gene results in a CD163 protein which cannot bind and/oruncoat a porcine reproductive and respiratory syndrome virus (PRRSV) iscan be produced by methods which are generally the same as thosedescribed in Example 1. First, a targeting vector is inserted into adonor cell, where it can recombine with the endogenous CD163 gene. Thedonor cells with this specific modification are then selected and usedfor somatic cell nuclear transfer to create a genetically modifiedembryo. The embryo is then transferred to a surrogate for termgestation. After identifying transgenic swine with an inactivated CD163allele by either PCR or Southern blotting, these animals are permittedto reach sexual maturity, and are then used for natural mating totransmit the gene to fetuses or offspring.

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When introducing elements of the present invention or the preferredembodiments(s) thereof, the articles “a”, “an”, “the” and “said” areintended to mean that there are one or more of the elements. The terms“comprising”, “including” and “having” are intended to be inclusive andmean that there may be additional elements other than the listedelements.

In view of the above, it will be seen that the several objects of theinvention are achieved and other advantageous results attained.

As various changes could be made in the above products and methodswithout departing from the scope of the invention, it is intended thatall matter contained in the above description and shown in theaccompanying drawing[s] shall be interpreted as illustrative and not ina limiting sense.

What is claimed is:
 1. A genetically modified swine comprising aninactivating mutation in an allele of a CD163 gene.
 2. The geneticallymodified swine of claim 1, wherein the swine comprises inactivatingmutations in both alleles of the CD163 gene.
 3. The genetically modifiedswine of claim 1, wherein the mutation alters the expression or activityof CD163.
 4. The genetically modified swine of claim 2, wherein themutations alter the expression or activity of CD163.
 5. The geneticallymodified swine of claim 1, wherein the mutation comprises an insertion,a deletion, a frame shift mutation, a nonsense mutation, a missensemutation, or a point mutation.
 6. The genetically modified swine ofclaim 2, wherein the mutations comprise insertions, deletions, frameshift mutations, nonsense mutations, missense mutations, pointmutations, or a combination thereof.
 7. The genetically modified swineof claim 1, wherein the mutation comprises a partial deletion or acomplete deletion of the allele.
 8. The genetically modified swine ofclaim 2, wherein the mutations comprise partial deletions of thealleles, complete deletions of the alleles, or a combination thereof. 9.The genetically modified swine of claim 1, wherein the mutationcomprises a complete knockout of the CD163 allele.
 10. The geneticallymodified swine of claim 2, wherein the mutations comprise completeknockouts of the CD163 alleles.
 11. The genetically modified swine ofclaim 1, wherein the inactivating mutation results in a CD163 proteinwhich cannot bind and/or uncoat a porcine reproductive and respiratorysyndrome virus (PRRSV).
 12. The genetically modified swine of claim 2,wherein at least one of the inactivating mutations results in a CD163protein which cannot bind and/or uncoat a porcine reproductive andrespiratory syndrome virus (PRRSV).
 13. A method for producing agenetically modified swine of claim 1, the method comprising: editingthe genetic content of a porcine cell to create a modification whichalters the expression or activity of CD163; and generating an animalfrom the cell.
 14. The method of claim 13, wherein the editing comprisesthe use of a zinc-finger nuclease or a TAL effector nuclease.
 15. Themethod of claim 13, wherein the editing results in partial or completedeletion of a CD163 allele.
 16. The method of claim 13, wherein theediting results in an animal that produces a CD163 protein which cannotbind and/or uncoat a porcine reproductive and respiratory syndrome virus(PRRSV).
 17. The method of claim 13, wherein the method furthercomprises: mating a female animal produced by the method of claim 13with a male animal produced by the method of claim 13 to produce F1progeny; and screening the F1 progeny to identify animals having amodification in both alleles of a CD163 gene.
 18. The method of claim17, wherein the screening comprises using a polymerase chain reaction(PCR) or Southern blot.
 19. A method for producing a geneticallymodified swine of claim 1, the method comprising: introducing asite-specific nuclease into a porcine cell, the nuclease being adaptedto bind to a target sequence in an allele of the CD163 gene; andincubating the cell under conditions that permit the nuclease to actupon the DNA at or near the target sequence and thereby inducerecombination, homology-directed repair, or non-homologous end joiningat or near the target site.
 20. The method of claim 19, wherein thesite-specific nuclease comprises a zinc-finger nuclease or a TALeffector nuclease.
 21. The method of claim 19, wherein introducing thenuclease into the cell results in partial or complete deletion of aCD163 allele.
 22. The method of claim 19, wherein introducing thenuclease into the cell results in a cell that produces a CD163 proteinwhich cannot bind and/or uncoat a porcine reproductive and respiratorysyndrome virus (PRRSV).
 23. The method of claim 19, wherein the methodfurther comprises generating an animal from the cell.
 24. The method ofclaim 23, wherein the method further comprises mating a female animalproduced by the method of claim 23 with a male animal produced by themethod of claim 23 to produce F1 progeny; and screening the F1 progenyto identify animals having a modification in both alleles of a CD163gene.
 25. The method of claim 24, wherein the screening comprises usinga polymerase chain reaction (PCR) or Southern blot.